Myelodysplastic Syndrome (MDS) is a premalignant disorder of hematopoietic stem cells. MDS is usually fatal unless treated with allogeneic bone marrow transplantation, an option not generally available for persons over age 60, who are most often afflicted by this disease. Despite recent advances in single nucleotide polymorphism analyses, in ~30% of MDS patients, chromosomal defects are indiscernible; here, these cases are termed MDS with normal karyotype (MDS-NK). The genes and pathways driving MDS-NK remain elusive. Therefore, to improve the current suboptimal therapeutic outcomes in MDS-NK, there is an urgent need to identify and validate therapeutic target genes and pathways involved in the progression of MDS-NK. The proliferation and differentiation defects in MDS-NK stem cells play a major role in determining the pace of progression and acquisition of more aggressive phenotypes including secondary acute myelogenous leukemia (sAML) and bone marrow failure. Since MDS-NK exhibit heterogeneous morphologies and clinical phenotypes, it may take considerable effort and time before linking the cryptic molecular lesions in the MDS- NK genome to the pathophysiology of this disease. To overcome this grim scenario, we propose a novel high- throughput, genome-wide, cell-based functional genomics screen called GRIP (genome-wide random insertion of promoter) to identify and validate the genes that drive MDS-NK pathogenesis. The GRIP method has the potential to achieve the activation of virtually any gene within the genome of stem cells. The fundamental hypothesis of this proposal is that elevated expression of gain-of-function genes that cause abnormal proliferation and differentiation of MDS stem cells promotes MDS-NK pathogenesis. We will test this hypothesis by completing two specific aims. By screening four GRIP libraries of one million MDS-NK stem cells each Aim 1 will discover and validate the drivers of MDS-NK in an in vitro (BFU-GEMM assays). By examining the correlation between the expression of the identified target genes and clinical outcomes in a retrospective clinical study, Aim 2 will define the clinical relevance of target genes identified from GRIP screening in driving MDS-NK. Major innovative aspects of this project are the ability of rapid identification of genes that drive MDS-NK pathogenesis in an unbiased, cell-based, functional genomics screen without the prior knowledge of gene sequence. Uncovering these genes will advance our understanding of how MDS-NK develops and progresses into high-risk MDS and sAML and provide targets for (a) therapies and/or (b) markers for prognoses. Additionally, uncovering of the genes involved in the proliferation and differentiation of MDS stem cells will advance the understanding of the biology of cancer-initiating stem cells. Our proposal constitutes a synergistic collaboration between two laboratories, one conducting basic science at Texas A&M University-Commerce and the other translational research at the Cleveland Clinic.